Catherine J. Wu, MD and David A. Reardon, MD Project 1: Personalized Neoantigen Vaccination and Anti-PD-1 Therapy for Glioblastoma Summary Current therapies for glioblastoma (GBM), the most common malignant primary brain cancer, remain palliative. These tumors have responded poorly to single agent immunotherapy approaches due to several factors including the fact that GBM is an immunologically cold tumor with a paucity of effector T cells infiltrating the tumor microenvironment. We have developed a personalized tumor neoantigen-targeting vaccine strategy (NeoVax) based on robust analytic sequencing pipelines and have demonstrated marked CD4+ and CD8+ T cell responses to vaccinated neoepitope peptides among patients with high-risk melanoma (Ott & Hu, Nature, 2017) and with newly diagnosed GBM (Reskin et al., Nature 2019). Furthermore, in collaboration with investigators from Project 3, we demonstrated that NeoVax induced a marked influx of intratumoral immune effector cells, including CD4+ T cells with specificity to an immunizing neoepitope into the GBM tumor microenvironment. We now extend this work by conducting a second trial in which NeoVax will be combined with anti-PD-1 therapy for GBM patients based on our observation that anti-PD-1 therapy broadened anti-tumor immune responses among our melanoma patients treated with NeoVax. We have designed our trial to address the key question whether timing of PD-1 blockade relative to neoantigen priming with NeoVax affects T cell memory responses, based on recent work by Dr. Sharpe (Project 2) demonstrating that PD-1 signaling critically regulates long-term T cell memory. We hypothesize anti-PD-1 therapy will improve outcome for GBM patients undergoing NeoVax therapy and that timing of PD-1 blockade relative to tumor neoantigen priming with NeoVax will critically influence the generation of polyfunctional, long-term memory T cell responses. We will evaluate these hypotheses in work organized into three specific aims. In Aim 1, we will evaluate the administration of PD-1 blockade either before or after NeoVax priming in a phase 1b trial for newly diagnosed GBM patients who are treated with standard radiotherapy. We will evaluate the strength, breadth and state of circulating neoantigen-specific T cell responses relative to timing of PD-1 blockade including long-term T cell memory responses among patients treated on this trial in Aim 2. We will similarly interrogate the composition and functional states of tumor infiltrating immune cells before and after NeoVax plus anti-PD-1 therapy in Aim 3. T cell immune responses for Aims 2 and 3 will be analyzed in Core 1, while Core 2 will perform the computational and statistical analyses of these data.